Mid-infrared spectroscopy (MIRS) is a well-established analytical tool for qualitative and quantitative analysis of soil samples. However, effects of soil sample grinding procedures on the prediction accuracy of MIR models and on qualitative spectral information have not been well investigated and, in consequence, not standardized up to now. Further, the effects of soil sample selection on the accuracy of MIR prediction models has not been quantified yet. This study investigated these effects by using 180 well-characterized soil samples that were ground for different times (0, 2 or 4 minutes) and then used for MIR measurements. To study the impact of sample preparation, soil spectra were subjected to principal component analyses (PCA), multiple regression and partial least square (PLS) analysis. The results indicate that the prediction accuracy of MIR models for soil organic carbon (SOC) and pH and the qualitative spectral information were better overall for lightly ground (2 minutes) soil samples compared with intensively (4 minutes) or unground soil samples. Whereas the grinding procedure did not show any effect on spectra of clay minerals, spectral information for quartz and for SOC was modified. Even though it is difficult to recommend a global standardized soil sample grinding procedure for MIR measurements because of different mill types available within laboratories, we highly recommend using an internally standardized grinding procedure. Moreover, we show that neither land use nor soil sampling depth influences the prediction of the SOC content. However, sand and clay content substantially affect the score vectors used by the PLS algorithm to predict the SOC content. Thus, we recommend using soil samples similar in texture for more precise SOC calibration models for MIR spectroscopy.